Abstract
Human AC133 antigen, also called CD133, was recently identified as a hematopoietic stem cell marker. However, the molecular structure and function of this protein has remained unclear. Here we cloned and identified a novel isoform of AC133, which we named AC133-2. In comparison to the reported AC133 cDNA, which is referred to herein as AC133-1, a small exon of 27 nucleotides is deleted in AC133-2 by alternative mRNA splicing. Similar to the previously characterized AC133 antigen, recombinant AC133-2 expressed in 293 cells was glycosylated and transported to plasma membrane. AC133-2 mRNA was found predominant in a variety of human fetal tissue, adult tissues, and several carcinomas. In contrast, AC133-1 mRNA was more prominent in fetal brain and adult skeletal muscle but was not detected in fetal liver and kidney, adult pancreas, kidney, and placenta, suggesting different roles for the two isoforms in fetal development and mature organ homeostasis. Here, we demonstrate that AC133-2 is the isoform expressed on hematopoietic stem cells derived from fetal liver, bone marrow, and peripheral blood. The results indicate that AC133-2, not AC133-1, has been the cell surface antigen recognized by anti-AC133 monoclonal antibodies that are used for isolation of hematopoietic stem cells. To further investigate its expression in other stem cell populations, we found that AC133-2 co-expressed with beta(1) integrin in the basal layer of human neonatal epidermis. AC133-2(+)/beta(1) integrin(+) cells proliferated and differentiated in culture, which coincided with a loss of AC133-2 and gain in a terminal differentiation marker involucrin. Taken together, these results suggest that AC133-2 is expressed in multiple stem cell niches and may provide a means to isolate specific stem cell subpopulations from human tissues.
Highlights
Human AC133 antigen is a glycoprotein with a molecular mass of ϳ120 kDa
The results indicate that AC133-2, not AC133-1, has been the cell surface antigen recognized by anti-AC133 monoclonal antibodies that are used for isolation of hematopoietic stem cells
Cloning and Identification of AC133-2—To make a Northern blot cDNA probe for human AC133 antigen, we amplified a N-terminal fragment of 458 bp (GenBankTM accession number AF027208) by RT-PCR from the retinoblastoma cell line WERIRB-1 from which the first AC133 cDNA was cloned [1]
Summary
Cell Lines and Reagents—Retinoblastoma cell line WERI-RB-1 and irradiated 3T3 mouse embryo cells were purchased from American Tissue Culture Collection (Manassas, VA). 293 cells were kindly provided by Dr Yashushi Numaguchi (Children’s Hospital, Boston, MA).
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